| Background: In order to alleviate the effect of ischemia reperfusion injury on organs in surgery,Ischemic Preconditioning(IPC)and Remote Ischemic Preconditioning(RIPC)were invented.However,the protective effects of RIPC were controversial and its mechanism and humoral factor needed further research.Researches suggested Hypoxia-Inducible Factor-1(HIF-1)and Adrenomedullin(ADM)might participate in RIPC’s effect.Thus,the aim of this study was to investigate whether RIPC offered protection to kidney suffering ischemia reperfusion and make the role of HIF-1 and ADM clear.Materials and Methods: Male Sprague-Dawley rats were applied in this study.The model of acute renal ischemia reperfusion was created in partⅠsection one.There were four groups in partⅠsection two,two of which were separately applied acute or delayed RIPC,and the protection difference and the expression of HIF-1 were compared.In partⅡ,groups were separately receiving inhibitior YC-1,agonist DMOG and ADM,the protection of RIPC and the expression of HIF-1 and ADM were observed.Results: 1.In partⅠsection one,the group which adopted the method of 45 min ischemia of the left kidney and the right harvested had a significant increase in its renal function indicators and oxidative damage indicators together with its tubular score.What’s more,the injury was increased in 24 h.2.In partⅠsection two,both the acute and delayed group had an improvement in renal function indicators and oxidative damage indicators,and the delayed one was better.Besides,there was an increased expression of HIF-1 and ADM in the delayed group.3.In partⅡ,the one received ADM had a better effect than the ischemia reperfusion group,but lower than delayed one.And the one received DMOG had a better protection than others.Conclusions: It was appropriate to construct the acute renal ischemia reperfusion injury model by applying the method of 45 min ischemia of the left kidney and the right harvested.It could offer protection,to some extent,to the renal function suffering acute ischemia reperfusion injury by applying the method via limb ischemic preconditioning.And the delayed phase of RIPC had a better protection.HIF-1 involved in RIPC’s protective effect and exogenous ADM could provide protection to renal function suffering ischemia reperfusion.The regulation mechanism of ADM in humoral pathway was complex,which needed further research.Part Ⅰ Section One:The Construction Of The Acute Ischemic Preconditioning ModelBackground: Acute kidney injury(AKD)was a kind of common clinical syndrome,which manifested a period of acute and rapid decline in renal function and accumulation of metabolic waste.AKI induced by acute ischemia reperfusion injury in surgery was also a common clinical postoperative complication,leading to the increase of mortality rate.Thus,constructing a proper model was the understructure of researching for acute renal ischemia reperfusion injury.The purpose of this study was to construct a proper rat model in combination with the preliminary experiment and the former studies.Materials and Methods: Male Sprague-Dawley rats were divided into two groups: 1.Sham group: Opening the abdomen,isolating both the left and the right sides of renal pedicles,then closing the abdomen.2.Acute ischemia reperfusion group(IR): Opening the abdomen,isolating both the left and the right sides of renal pedicles,occluding the left renal pedicle for 45 min by applying the minimally invasively vascular clamp while removing the right kidney,then loosing the clamp,closing the abdomen.At the point of 6 h and 24 h after operation,animals were sacrificed and samples harvested.Creatinine(Cr),blood urea nitrogen(BUN),myeloperoxidase(MPO),malondialdehyde(MDA)and superoxide dismutase(SOD)were measured,and hematoxylin-eosin staining(HE)was done to evaluate the histopathological changes together with the tubular score.Results: 1.In the two point,the results of renal function indicators Cr,BUN and the oxidative damage indicators MPO,MDA,SOD in IR group were significantly increasing than the one in sham group,indicating deterioration of the renal injury(P<0.001).2.The renal injury was deteriorating in the 24 h after operation and reached a high point.Conclusions: It was appropriate to construct the acute renal ischemia reperfusion injury model by applying the method of 45 min ischemia of the left kidney and the right harvested.Part Ⅰ Section Two:The Comparison Of The Protective Effect Offered By Remote Ischemic Preconditioning To Renal Function Suffering Ischemia Reperfusion InjuryBackground: During urological surgery such as partial nephrectomy,kidney transplantation and so on,kidney would suffer a period of ischemia which led to the ischemia reperfusion injury.In order to decrease the injury,many protective measures such as ischemic preconditioning(IPC)were created.IPC was initially applied in cardiovascular system to protect heart.For the purpose of avoiding the injury on blood vessel induced by IPC,an update version remote ischemic preconditioning(RIPC)came out.RIPC could be applied to non-cardiac organs and there was significant difference between the protections offered by different phase of RIPC-acute phase and delayed phase.In urology,the application of RIPC was on an initial stage,and there was still controversy in the renal protection offered by RIPC.And in the research of the RIPC’s mechanism,it was found hypoxia-inducible factor-1(HIF-1)might involve in it.Thus,the purpose of this section was to investigate the protective effective offered by RIPC to renal function suffering ischemia reperfusion and the effect of HIF-1 involving in RIPC.Materials and Methods: Male Sprague-Dawley rats were divided into four groups: 1.Sham group: Opening the abdomen,isolating both the left and the right sides of renal pedicles,then closing the abdomen.2.Acute ischemia reperfusion group(IR): Opening the abdomen,isolating both the left and the right sides of renal pedicles,occluding the left renal pedicle for 45 min by applying the minimally invasively vascular clamp while removing the right kidney,then loosing the clamp,closing the abdomen.3.Acute RIPC group(Acute): Receiving RIPC and after that taking measures as IR group instantaneously.4.Delayed RIPC group(Delayed): Receiving RIPC and taking measures as IR group 24 h later.At the point of 24 h after operation,animals were sacrificed and samples harvested.Creatinine(Cr),blood urea nitrogen(BUN),myeloperoxidase(MPO),malondialdehyde(MDA)and superoxide dismutase(SOD) were measured,and hematoxylin-eosin staining(HE)was done to evaluate the histopathological changes together with the tubular score.Besides,the expression of HIF-1α was measured by Western Blot and immunohistochemistry,and the level of ADM in blood was also measured.Results: 1.It was suggested that Acute and Delayed group were better than IR group the results of renal function indicators Cr,BUN and the oxidative damage indicators MPO,MDA,SOD and tubular score(P<0.05),indicating protection offered by RIPC led to the lower damage of kidney.2.The Delayed group was better than the Acute in results of BUN,MPO and tubular score,meaning the Delayed offered better protection than the Acute.3.After remote ischemic preconditioning,the expression of HIF-1α in Acute and Delayed group was increasingly higher than the IR group(P<0.05),and it was more expressed in Delayed than Acute(P<0.05).Moreover,the level of ADM in blood was significantly increased in Delayed group than the rest three(P<0.001).Conclusions: It could offer protection,to some extent,to the renal function suffering acute ischemia reperfusion injury by applying the method,3 ×(5I /5R)via limb ischemic preconditioning.There was a significance in the comparison with the protection offering by different phase of RIPC.And the delayed phase of RIPC had a better protection than the acute phase.HIF-1 and ADM might participate in RIPC’s protection mechanism.Part Ⅱ:The Mechanism Of HIF-1 And ADM in Remote Ischemic Preconditioning Offering Protection To Renal Function Suffering Ischemia ReperfusionBackground: Remote ischemic preconditioning could offer protection to kidney suffering ischemia reperfusion.Though the mechanism of RIPC was still not clear,it was known that it was consisted of three pathway: 1.Neuronal pathway.2.Systemic response.3.Humoral pathway.In humoral pathway the humoral factor which conveyed the preconditioning signal from the remote to the target had not been identified yet.In preliminary study,it manifested RIPC offered protection to renal function,and the delayed phase had better effect.Besides,hypoxia-inducible factor-1α was highly expressed after RIPC.And the level of ADM in blood was also increased in delayed RIPC.Considering HIF-1 and ADM might involve in RIPC,the purpose of this part was to investigate HIF-1α and ADM’s effect in RIPC and whether ADM was the candidate in humoral pathway.Materials and Methods: Male Sprague-Dawley rats were divided into six groups: 1.Sham group: Opening the abdomen,isolating both the left and the right sides of renal pedicles,then closing the abdomen.2.Acute ischemia reperfusion group(IR): Opening the abdomen,isolating both the left and the right sides of renal pedicles,occluding the left renal pedicle for 45 min by applying the minimally invasively vascular clamp while removing the right kidney,then loosing the clamp,closing the abdomen.3.Delayed RIPC group(Delayed): Receiving RIPC and taking measures as IR group 24 h later.4.YC-1 group: Injecting YC-1 2 h before RIPC,then taking the same measures as Delayed group.5.DMOG group: Injecting DMOG 2 h before RIPC,then taking the same measures as Delayed group.6.ADM group: Injecting ADM 2 h before RIPC,then taking the same measures as IR group.The sham,IR,Delayed group received vehicle at the same time point.At the point of 24 h after operation,animals were sacrificed and samples harvested.Creatinine(Cr),blood urea nitrogen(BUN),myeloperoxidase(MPO),malondialdehyde(MDA)and superoxide dismutase(SOD) were measured,and hematoxylin-eosin staining(HE)was done to evaluate the histopathological changes together with the tubular score.Besides,the expression of HIF-1α was measured by Western Blot and immunohistochemistry,and the level of ADM in blood and renal tissue was also measured by ELISA and IHC.Results: 1.With the help of drug intervention,the protection offered by the three delayed RIPC group differed with each other.The renal function indicators Cr and BUN in Delayed and DMOG group were better than the IR one(P<0.01).After receiving exogenous ADM,there was no significance between ADM and IR group(P>0.05).What’s more,the protection offered by ADM group in the oxidative damage indicators was lower than Delayed and DMOG group.2.With the help of drug intervention,there was a significant difference in the expression of HIF-1α in each group,among which it was higher in Delayed,DMOG and YC-1 than ADM group(P < 0.05).3.After receiving exogenous ADM,the expression of ADM in ADM group was higher than IR group,but lower than the Delayed and the DMOG(P<0.05).4.Though the protection offered by ADM group was significantly weakened than Delayed group,it had fewer renal injury and lower tubular score than YC-1group(P<0.05).Conclusions: HIF-1 involved in RIPC’s protective effect and increasing or decreasing the expression of HIF-1 could enhance or weaken RIPC’s effect.Exogenous ADM could provide protection to renal function suffering ischemia reperfusion.The expression and effects of HIF-1 and ADM were related to the protection of RIPC.The regulation mechanism of ADM in humoral pathway was complex,which may be related to the expression of HIF-1.As a protective factor in RIPC,the protective mechanism of ADM needed further research. |
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